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Patent 2703102 Summary

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Claims and Abstract availability

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(12) Patent: (11) CA 2703102
(54) English Title: DEPTH OF FIELD EXTENSION FOR OPTICAL TOMOGRAPHY
(54) French Title: EXTENSION DE LA PROFONDEUR DE CHAMP POUR TOMOGRAPHIE OPTIQUE
Status: Granted
Bibliographic Data
(51) International Patent Classification (IPC):
  • G02F 1/01 (2006.01)
(72) Inventors :
  • RAHN, RICHARD J. (United States of America)
  • HAYENGA, JON W. (United States of America)
(73) Owners :
  • VISIONGATE, INC. (United States of America)
(71) Applicants :
  • VISIONGATE, INC. (United States of America)
(74) Agent:
(74) Associate agent:
(45) Issued: 2016-11-29
(86) PCT Filing Date: 2008-10-22
(87) Open to Public Inspection: 2009-04-30
Examination requested: 2013-10-04
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2008/080726
(87) International Publication Number: WO2009/055429
(85) National Entry: 2010-04-20

(30) Application Priority Data:
Application No. Country/Territory Date
11/876,658 United States of America 2007-10-22

Abstracts

English Abstract




An optical projection tomography system is illuminated with a light source
(130). An object-containing tube (107),
a portion of which is located within the region illuminated by the light
source (130), contains an object of interest (114) that has a
feature of interest. A detector (112) is located to receive emerging radiation
from the object of interest (114). A lens (103), including
optical field extension elements (1), is located in the optical path between
the object region and the detector (104), such that light
rays (130) from multiple object planes (Zn) in the object-containing tube
(107) simultaneously focus on the detector (104). The
object-containing tube (107) moves relatively to the detector (104) and the
lens (103) operate to provide multiple views of the object
region for producing an image of the feature of interest at each view.


French Abstract

Un système de tomographie par projection optique est éclairé avec une source de lumière (130). Un tube contenant un objet (107), dont une partie est située à l'intérieur de la région éclairée par la source de lumière (130), contient un objet d'intérêt (114) qui comporte une caractéristique d'intérêt. Un capteur (112) est situé pour recevoir un rayonnement émergent en provenance de l'objet d'intérêt (114). Un objectif (103) comprenant des éléments d'extension de champ optique (1) est situé dans le trajet optique entre la région de l'objet et le capteur (104) de telle sorte que les rayons de lumière (130) provenant de multiples plans (Zn) de l'objet situé dans le tube contenant l'objet (107) se focalisent simultanément sur le capteur (104). Le tube contenant l'objet (107) se déplace par rapport au capteur (104) et l'objectif (103) fonctionne pour donner de multiples vues de la région d'intérêt pour produire une image de la caractéristique d'intérêt pour chaque vue.

Claims

Note: Claims are shown in the official language in which they were submitted.


CLAIMS:
1. An optical projection tomography system for imaging an object of
interest, the optical projection tomography system comprising:
(a) a light source;
(b) an object-containing tube, a portion of which is located within the
region illuminated by the light source, wherein the object of interest has at
least one
feature of interest located within the object-containing tube;
(c) at least one detector, wherein the at least one detector is located to
receive emerging radiation from the object of interest; and
(d) at least one lens located in the optical path between the object
region and the at least one detector, wherein the at least one lens includes
at least
one optical field extension element such that light rays from multiple object
planes in
the object-containing tube simultaneously focus on the at least one detector.
2. The system of claim 1 wherein the object moves relatively to the at
least
one detector and the at least one lens operates to provide multiple views of
the object
region for producing at least one image of the at least one feature of
interest at each
view.
3. The system of claim 1, wherein the object of interest comprises a
biological cell.
4. The system of claim 1, wherein each of the multiple object planes that
come to a focus on at least one of the at least one detectors registers light
of a
different optical wavelength on the at least one detector.
5. The system of claim 1, wherein an interval spanned by the multiple
object planes comprises an interval spanning the thickness of the object of
interest.
27

6. The system of claim 1, wherein the object of interest is stained to
impart
an absorption coefficient of at least one wavelength that registers on the at
least one
detector.
7. The system of claim 1, wherein an interval spanned by the multiple
object planes comprises an interval spanning the thickness of the diameter of
the
object-containing tube.
8. The system of claim 1, wherein the system further comprises a
chromatic filter array located between the at least one lens and the at least
one
detector.
9. The system of claim 1, wherein the system further comprises a
piezoelectric transducer coupled to the at least one lens for moving the at
least one
lens along an optical path between each of the multiple views of the object
region,
such that the object of interest remains within an interval spanned by the
multiple
object planes.
10. The system of claim 1, further comprising a wavefront-coded optical
element located between the at least one lens and the at least one detector.
11. The system of claim 1 wherein the at least one detector comprises a
detector selected from the group consisting of charge coupled devices,
complementary metal-oxide-semiconductor devices, solid-state image sensors,
and
solid-state image sensor detector arrays.
12. The system of claim 6, wherein the object of interest is stained with
hematoxylin.
13. The system of claim 1 , wherein the light from multiple object planes
includes light having a wavelength range of 550 nm to 620 nm spanning a focus
interval of up to 50 microns.
28

14. The system of claim 1, wherein the object-containing tube has a
diameter of at least 50 microns.
15. The system of claim 2, wherein the system further comprises a
piezoelectric transducer attached to the at least one lens for moving the at
least one
lens along an optical path between each of the multiple views of the object
region,
such that the object of interest remains within the focus interval.
16. The system of claim 1, wherein the system further comprises a
chromatic filter array located between the at least one lens and the at least
one
detector.
17. The system of claim 1, wherein an interval spanned by the multiple
object planes comprises a focus interval of at least 12 microns.
18. The system of claim 1, wherein the light from multiple object planes
includes a wavelength range of 550 nm to 620 nm.
19. The system of claim 1, further comprising a band pass filter for
passing
light in the range of 550 nm to 620 nm located to band limit light reaching
the at least
one detector.
20. The system of claim 1, wherein the light source includes a chromatic
filter located to filter light reaching the object-containing tube.
21. The system of claim 1, wherein the at least one lens comprises a
hyperchromatic lens.
22. An optical tomography system for viewing an object of interest
comprising:
a microcapillary tube viewing area for positioning the object of interest;
at least one detector;
29

a motor located to attach to and rotate a microcapillary tube;
means for transmitting broadband light having wavelengths between
550 nm and 620 nm into the microcapillary tube viewing area;
a hyperchromatic lens located to receive light transmitted through the
microcapillary tube viewing area; and
a tube lens located to focus light rays transmitted through the
hyperchromatic lens, such that light rays from multiple object planes in the
microcapillary tube viewing area simultaneously focus on the at least one
detector.
23. The system of claim 22, wherein the object of interest comprises a
biological cell.
24. The system of claim 22, wherein the hyperchromatic lens and the tube
lens operate to simultaneously focus multiple object planes from the
microcapillary
tube viewing area on the at least one detector.
25. The system of claim 24, wherein an interval spanned by the multiple
object planes comprises an interval spanning the thickness of the biological
cell.
26. The system of claim 23, wherein the biological cell is stained to
impart
an absorption coefficient of at least one wavelength that registers on the at
least one
detector.
27. The system of claim 23, wherein an interval spanned by the multiple
object planes comprises an interval spanning the thickness of the
microcapillary tube
viewing area.
28. The system of claim 22, wherein the system further comprises a
chromatic filter array located between the hyperchromatic lens and the at
least one
detector.

29. The system of claim 22, wherein the light from the multiple object
planes includes light having a wavelength range of 550 nm to 620 nm spanning a

focus interval of up to 50 microns.
30. The system of claim 22, further comprising a chromatic filter array
located between the hyperchromatic lens and the at least one detector, so that
light
coming to a focus on the detector is separated into two or more wavelength
bands,
each wavelength band being transmitted through the chromatic filter array to a

separate set of pixels on the at least one detector.
31. An optical tomography system for viewing an object of interest
comprising:
a microcapillary tube containing the object of interest;
a motor, attached to the microcapillary tube, for rotating the
microcapillary tube;
a light source located to illuminate the microcapillary tube;
a hyperchromatic lens located to receive light transmitted through the
microcapillary tube;
a dichroic beamsplitter located to split a plurality of ray paths originating
in multiple object planes in the microcapillary tube as transmitted through
the
hyperchromatic lens; and
at least two detectors, where a first detector of the at least two detectors
is located to receive light transmitted along one of the plurality of ray
paths, and a
second detector of the at least two detectors is located to receive light
transmitted
along another of the plurality of ray paths, where light rays from the
multiple object
planes are simultaneously focused on at least one of the at least two
detectors.
31

32. The system of claim 31, wherein the system further comprises a
piezoelectric transducer coupled to the hyperchromatic lens.
33. The system of claim 31, wherein the object of interest comprises a
biological cell.
34. The system of claim 31, wherein each of the multiple object planes that

come to a focus registers light of a different optical wavelength on at least
one of the
at least two detectors.
35. The system of claim 31, wherein an interval spanned by the multiple
object planes comprises an interval spanning the thickness of the object of
interest.
36. The system of claim 31, wherein the biological cell is stained to
impart
an absorption coefficient of at least one wavelength.
37. The system of claim 31, wherein an interval spanned by the multiple
object planes comprises an interval spanning the thickness of the
microcapillary tube.
38. The system of claim 31, wherein the system further comprises a
chromatic filter array located between the hyperchromatic lens and each of the
at
least two detectors.
39. The system of claim 36, wherein the object of interest is stained with
hematoxylin.
40. The system of claim 31, wherein the light from multiple object planes
includes light having a wavelength range of 550 nm to 620 nm covering a focus
interval of up to 50 microns.
41. The system of claim 40, wherein the object-containing tube has a
diameter of at least 50 microns.
42. The system of claim 40, wherein an interval spanned by the multiple
object planes comprises a focus interval of at least 12 microns.
32

43. The system of claim 31, wherein the light from multiple object planes
includes a wavelength range of 550 nm to 620 nm.
44. The system of claim 31, further comprising a band pass filter for
passing light in the range of 550 nm to 620 nm located to band limit light
reaching the
at least two detectors.
45. The system of claim 31, wherein the light source includes a chromatic
filter located to filter light reaching the object-containing tube.
46. A folded system for optical tomography comprising:
an objective lens for transmitting collimated light;
a first dichroic beam-splitting cube located downstream from the
objective lens to split the collimated light transmitted by the objective lens
into a first
arm and a second arm;
the first arm has a wavelength and originates in a first focal plane;
a first tube lens is positioned to transmit the first arm through a second
beam-splitter cube, and onto a first region of the camera sensor's active
area;
the second arm has a wavelength and originates in a second focal
plane;
first and second mirrors positioned to pass the second arm through a
tube lens after reflecting within a second dichroic beam-splitter cube and
onto a
second region of a camera sensor's active area, whereby the first region and
the
second region of the camera acquire focused images originating from different
focal
planes in object space.
47. The system of claim 46 where a relative lateral shift in images is
achieved by laterally shifting the second dichroic beam-splitter cube, so that
the
33

reflected light of the second arm is laterally shifted relative to the first
arm and to the
first tube lens.
48. The system of claim 1, wherein the at least one optical field
extension
element comprises a polarizing filter array located between the at least one
lens and
the at least one detector.
49. The system of claim 1, wherein the at least one optical field
extension
element comprises a wavefront-coding element located between the
hyperchromatic
lens and each of the at least two detectors.
50. The system of claim 1, wherein each of the multiple object planes that
come to a focus on the at least one detector is formed by light having a
different
polarization.
51. An optical projection tomography method for imaging an object of
interest, the optical projection tomography method comprising:
(a) illuminating an object of interest; and
(b) simultaneously focusing light rays from multiple object planes in the
object of interest onto at least one detector for a plurality of different
views.
52. The method of claim 51 further comprising filtering the light rays to
pass
only light rays having wavelengths within the range of 550 nm to 620 nm.
53. The method of claim 52 wherein the filtering further comprises passing
the light through a band pass filter located to limit light reaching at least
one detector.
54. The method of claim 51 further comprising delivering the object of
interest for imaging from an object containing tube; wherein the object
containing
tube comprises a tube selected from the group consisting of a capillary tube,
a
microcapillary tube, a microcapillary tube bundle, and a microcapillary tube
cassette.
34

55. The method of claim 54 wherein the object containing tube has a
diameter of at least 50 microns.
56. The method of claim 51, wherein simultaneously focusing comprises
moving at least one lens of a hyperchromatic lens system relative to at least
one
detector.
57. The method of claim 51 further comprising splitting a first plurality
of
light ray paths via a beamsplitter.
58. The method of claim 51 wherein the simultaneous focusing comprises
focusing a hyperchromatic lens.
59. The method of claim 56 wherein the simultaneous focusing further
comprises filtering the light by transmitting the light through at least one
chromatic
filter array.
60. The method of claim 51 wherein the at least one detector comprises a
detector selected from the group consisting of charge coupled devices,
complementary metal-oxide-semiconductor devices, solid-state image sensors,
and
solid-state image sensor detector arrays.
61. The method of claim 59, further comprising locating the at least one
chromatic filter array between the hyperchromatic lens and the at least one
detector,
so that light coming to a focus on the detector is separated into two or more
wavelength bands, each wavelength band being transmitted through the chromatic

filter array to a separate set of pixels on the at least one detector.
62. The method of claim 59, wherein the at least one chromatic filter array

is substituted with at least one polarization filter array.
63. The method of claim 51, wherein simultaneously focusing of the
multiple object planes comprises extending focusing through a focus interval
spanning the thickness of the object of interest.

64. The method of claim 51, wherein the simultaneously focusing of the
multiple object planes comprises extending focusing through a focus interval
of at
least 12 microns.
65. The method of claim 51, wherein the simultaneously focusing of the
multiple object planes comprises extending focusing through a focus interval
of up to
50 microns.
66. The method of claim 51 further comprising impinging the light rays onto

a wave front-coded optical element.
67. The method of claim 51, further comprising staining the object of
interest to impart an absorption coefficient of at least one wavelength.
68. The method of claim 67, wherein the staining of the object of interest
is
performed with hematoxylin.
69. The method of claim 51, wherein simultaneously focusing of the
multiple object planes comprises simultaneously focusing with an autofocusing
system.
70. The method of claim 66, wherein the simultaneously focusing with an
autofocusing system comprises focusing using chromatic balance.
71. The method of claim 51, wherein simultaneously focusing light rays
comprises transmitting the light rays through a polarizing filter array.
72. The method of claim 51, wherein simultaneously focusing light rays
comprises transmitting the light rays through a wavefront-coding element.
73. The method of claim 51, wherein simultaneously focusing light rays
comprises focusing each of the multiple object planes on the at least one
detector by
light having a different polarization.
36

74. The method of claim 51, wherein simultaneously focusing light rays
comprises performing a 2.5-D focus evaluation to determine a best focus.
75. The method of claim 51, wherein the object of interest comprises a
biological cell.
37

Description

Note: Descriptions are shown in the official language in which they were submitted.


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DEPTH OF FIELD EXTENSION FOR OPTICAL TOMOGRAPHY
Technical Field
The present invention relates to optical tomographic imaging systems in
general, and, more particularly, to optical projection tomography, in which a
small
object, such as a biological cell, is positioned in a capillary tube for
imaging by a
microscope.
Background
Advances in imaging biological cells using optical tomography have been
developed by Nelson as disclosed, for example, in US Patent No. 6,522,775,
issued
2/18/2003, and entitled "Apparatus and method for imaging small objects in a
flow
stream using optical tomography ".
Further developments in the field are taught in Fauver et at., US Patent
application number 10/716,744, filed 11/18/2003 and published as US
Publication
No. US-2004-0076319-A1 on 4/22/2004, entitled "Method and apparatus of
shadowgram formation for optical tomography" (Fauver '744) and Fauver et al.,
US
Patent application number 11/532,648, filed 9/18/2006, entitled "Focal plane
tracking
for optical microtomography " (Fauver '648).
Processing in such an optical tomography system begins with specimen
preparation. Typically, specimens taken from a patient are received from a
hospital
or clinic and processed to remove non-diagnostic elements, fixed and then
stained.
Stained specimens are then mixed with an optical gel, inserted into a micro-
capillary
tube and images of objects, such as cells, in the specimen are produced using
an
optical tomography system. The resultant images comprise a set of extended
depth
of field images from differing perspectives called "pseudo-projection images."
The
set of pseudo-projection images can be reconstructed using backprojection and
filtering techniques to yield a 3D reconstruction of a cell of interest.
The 3D reconstruction then remains available for analysis in order to enable
the quantification and the determination of the location of structures,
molecules or
molecular probes of interest. An object such as a biological cell may be
labeled with
at least one stain or tagged molecular probe, and the measured amount and
location
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of this probe may yield important information about the disease state of the
cell,
including, but not limited to, various cancers such as lung, breast, prostate,
cervical
and ovarian cancers.
In Optical Projection Tomography Microscopy (OPTM) systems as described,
for example, in Fauver '648, about 250 sample images taken over a 180-degree
rotation are required to adequately sample the volume of a cell nucleus
randomly
distributed in a flow stream within a 50 micron capillary tube.
The present disclosure provides new and novel techniques for providing
higher resolution and improved signal to noise ratio in order to reduce
sampling
requirements while maintaining acceptable resolution.
In one type of optical tomography system, as described in Fauver '744 and
constructed by VisionGate, Inc., the depth of field of the imaging optics is
extended
by scanning an objective lens transverse to a capillary tube containing a
specimen. A
piezoelectric transducer (PZT) actuator transversely moves the objective lens
sinusoidally several times per second in order to scan a series of focal
planes though
a specimen. By using a PZT actuator to move the objective lens, a focal plane
moving through the specimen has its speed limited by inertia inherent in
moving the
objective lens mass rapidly along the optical axis through the specimen.
Typically,
an upper limit of the scan rate is roughly 60 cycles per second. With well-
synchronized rotation and objective scanning, an image can be acquired on the
down-stroke as well as the up-stroke of the PZT actuator, allowing up to 120
images
per second to be acquired. While this is a useful acquisition rate, it can be
significantly improved through the apparatus, systems and methods disclosed
herein.
Brief Summary of the Disclosure
An optical projection tomography system is illuminated with a light source. An

object-containing tube, a portion of which is located within the region
illuminated by
the light source, contains at least one object of interest that has at least
one feature
of interest. At least one detector is located to receive emerging radiation
from the
object of interest. A lens, including optical field extension elements, is
located in the
optical path between the object region and the detector, such that light rays
from
multiple object planes in the object-containing tube simultaneously focus on
the at
least one detector. The object-containing tube moves relatively to the at
least one
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detector and the lens operate to provide multiple views of the object region
for
producing at least one image of the at least one feature of interest at each
view.
According to one aspect of the present invention, there is provided an
optical projection tomography system for imaging an object of interest, the
optical
projection tomography system comprising: (a) a light source; (b) an object-
containing
tube, a portion of which is located within the region illuminated by the light
source,
wherein the object of interest has at least one feature of interest located
within the
object-containing tube; (c) at least one detector, wherein the at least one
detector is
located to receive emerging radiation from the object of interest; and (d) at
least one
lens located in the optical path between the object region and the at least
one
detector, wherein the at least one lens includes at least one optical field
extension
element such that light rays from multiple object planes in the object-
containing tube
simultaneously focus on the at least one detector.
According to another aspect of the present invention, there is provided
an optical tomography system for viewing an object of interest comprising: a
microcapillary tube viewing area for positioning the object of interest; at
least one
detector; a motor located to attach to and rotate a microcapillary tube; means
for
transmitting broadband light having wavelengths between 550 nm and 620 nm into

the microcapillary tube viewing area; a hyperchromatic lens located to receive
light
transmitted through the microcapillary tube viewing area; and a tube lens
located to
focus light rays transmitted through the hyperchromatic lens, such that light
rays from
multiple object planes in the microcapillary tube viewing area simultaneously
focus on
the at least one detector.
According to still another aspect of the present invention, there is
provided an optical tomography system for viewing an object of interest
comprising: a
microcapillary tube containing the object of interest; a motor, attached to
the
microcapillary tube, for rotating the microcapillary tube; a light source
located to
illuminate the microcapillary tube; a hyperchromatic lens located to receive
light
transmitted through the microcapillary tube; a dichroic beamsplitter located
to split a
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plurality of ray paths originating in multiple object planes in the
microcapillary tube as
transmitted through the hyperchromatic lens; and at least two detectors, where
a first
detector of the at least two detectors is located to receive light transmitted
along one
of the plurality of ray paths, and a second detector of the at least two
detectors is
located to receive light transmitted along another of the plurality of ray
paths, where
light rays from the multiple object planes are simultaneously focused on at
least one
of the at least two detectors.
According to yet another aspect of the present invention, there is
provided a folded system for optical tomography comprising: an objective lens
for
transmitting collimated light; a first dichroic beam-splitting cube located
downstream
from the objective lens to split the collimated light transmitted by the
objective lens
into a first arm and a second arm; the first arm has a wavelength and
originates in a
first focal plane; a first tube lens is positioned to transmit the first arm
through a
second beam-splitter cube, and onto a first region of the camera sensor's
active area;
the second arm has a wavelength and originates in a second focal plane; first
and
second mirrors positioned to pass the second arm through a tube lens after
reflecting
within a second dichroic beam-splitter cube and onto a second region of a
camera
sensor's active area, whereby the first region and the second region of the
camera
acquire focused images originating from different focal planes in object
space.
According to a further aspect of the present invention, there is provided
an optical projection tomography method for imaging an object of interest, the
optical
projection tomography method comprising: (a) illuminating an object of
interest; and
(b) simultaneously focusing light rays from multiple object planes in the
object of
interest onto at least one detector for a plurality of different views.
Brief Description of the Drawings
FIG. 1 schematically shows an example of a design for a
hyperchromatic optical lens system.
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FIG. 2A-FIG.2D schematically show qualitative examples of simulated
monochromatic modulation transfer functions (MTFs) for each of four
wavelengths,
as they might be evaluated at four separate image planes.
FIG. 3A-FIG. 3D schematically show qualitative examples of simulated
polychromatic modulation transfer functions over a range of wavelengths, as
they
might be evaluated at four separate image planes.
FIG. 4A schematically shows a detail of the object space at one viewing
angle in an optical tomography system incorporating a hyperchromatic optical
lens.
FIG. 4B schematically shows the operation of the optical tomography
system depicted schematically in FIG 4A.
FIG. 4C schematically shows a detail of the object space at a second
viewing angle in an optical tomography system incorporating a hyperchromatic
optical
lens.
FIG. 4D schematically shows the operation of the optical tomography
system depicted schematically in FIG 4C.
FIG. 5 schematically shows an example of a design for a chromatic filter
array.
FIG. 6A schematically illustrates a first viewing angle for a
hyperchromatic optical tomography system incorporating a chromatic filter
array.
FIG. 6B schematically illustrates a first viewing angle for a
hyperchromatic optical tomography system incorporating a chromatic filter
array.
FIG. 7A schematically illustrates object space for a first viewing angle
for a hyperchromatic optical tomography system incorporating a long depth of
field.
FIG. 7B schematically illustrates a first viewing angle for a
hyperchromatic optical tomography system incorporating a long depth of field.
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FIG. 7C schematically illustrates the object space for a second viewing
angle for a hyperchromatic optical tomography system incorporating a long
depth of
field.
FIG. 7D schematically illustrates a second viewing angle for a
hyperchromatic optical tomography system incorporating a long depth of field.
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FIG. 8A schematically illustrates a first viewing angle for a hyperchromatic
optical tomography system incorporating multiple detection paths.
FIG. 8B schematically illustrates a second viewing angle for a hyperchromatic
optical tomography system incorporating multiple detection paths.
FIG. 9 illustrates an example of focus invariance in an MTF range.
FIG. 10 shows a block diagram of a method for finding the midpoint of the
focus-invariant region in a focus-invariant optical tomography system.
FIG. 11A shows a block diagram of another method for finding the midpoint of
the focus-invariant region in a focus-invariant optical tomography system.
FIG. 11B shows a block diagram of yet another method for finding the
midpoint of the focus-invariant region in a focus-invariant optical tomography
system.
FIG. 12 schematically depicts an embodiment of an autofocusing system
using chromatic balance.
FIG. 13 shows another method for finding the midpoint of the focus-invariant
region in a focus-invariant optical tomography system, using two photo-diodes
with
spatial-frequency filtering.
FIG. 14 schematically shows a block diagram of a method for 2.5-D imaging
in a focus-invariant optical tomography system.
FIG. 15 illustrates an example of a folded optical system allowing
simultaneous imaging of two focal planes on a single camera.
FIG. 16 schematically illustrates a multiple-camera device for acquiring a
range of focal planes in an optical tomography system.
FIG. 17 illustrates a schematic diagram of an OPTM system including
wavefront coded optics.
In the drawings, identical reference numbers identify similar elements or
components. The sizes and relative positions of elements in the drawings are
not
necessarily drawn to scale. For example, the shapes of various elements and
angles
are not drawn to scale, and some of these elements are arbitrarily enlarged
and
positioned to improve drawing legibility. Further, the particular shapes of
the
elements as drawn, are not intended to convey any information regarding the
actual
shape of the particular elements, and have been solely selected for ease of
recognition in the drawings.
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Detailed Description of the Preferred Embodiments
The following disclosure describes several embodiments and systems for
imaging an object of interest. Several features of methods and systems in
accordance with example embodiments of the invention are set forth and
described
in the Figures. It will be appreciated that methods and systems in accordance
with
other example embodiments of the invention can include additional procedures
or
features different than those shown in Figures. Example embodiments are
described
herein with respect to biological cells. However, it will be understood that
these
examples are for the purpose of illustrating the principals of the invention,
and that
the invention is not so limited.
Additionally, methods and systems in accordance with several example
embodiments of the invention may not include all of the features shown in
these
Figures. Throughout the Figures, like reference numbers refer to similar or
identical
components or procedures.
Unless the context requires otherwise, throughout the specification and claims

which follow, the word "comprise" and variations thereof, such as, "comprises"
and
"comprising" are to be construed in an open, inclusive sense that is as
"including, but
not limited to."
Reference throughout this specification to "one example" or "an example
embodiment," "one embodiment," "an embodiment" or various combinations of
these
terms means that a particular feature, structure or characteristic described
in
connection with the embodiment is included in at least one embodiment of the
present invention. Thus, the appearances of the phrases "in one embodiment" or
"in
an embodiment" in various places throughout this specification are not
necessarily all
referring to the same embodiment. Furthermore, the particular features,
structures,
or characteristics may be combined in any suitable manner in one or more
embodiments.
Definitions
Generally as used herein the following terms have the following meanings
when used within the context of optical microscopy processes:
"Capillary tube" has its generally accepted meaning and is intended to include

transparent microcapillary tubes and equivalent items with an inside diameter
of 100 microns or less.

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"Depth of field" is the length along the optical axis within which the focal
plane
may be shifted before an unacceptable image blur is produced.
"Object" means an individual cell or other entity.
"Pseudo-projection" includes a single image representing a sampled volume
of extent larger than the native depth of field of the optics.
"Specimen" means a complete product obtained from a single test or
procedure from an individual patient (e.g., sputum submitted for analysis, a
biopsy, or a nasal swab). A specimen may be composed of one or more
objects. The result of the specimen diagnosis becomes part of the case
diagnosis.
"Sample" means a finished cellular preparation that is ready for analysis,
including all or part of an aliquot or specimen.
Chromatic Aberration Depth of Field Extension
Most simple lenses will produce wavelength-dependent focal positions known
as chromatic focal shift. Chromatic aberrations are typically undesirable in a
lens.
However, for a sufficiently broad absorption spectrum in a biological sample,
the
dispersion of chromatic aberration can in effect extend the depth of field
image of an
absorptive object or feature.
Wavelength-dependent lens material will produce a lens with chromatic
aberrations. Nearly all lens materials can have both positive and negative
index
shifts with wavelength. Lens designers typically choose lens materials to
compensate for the chromatic focal plane shifts, resulting in a net chromatic
focal
shift near zero. For an example of an immersion microscope objective which is
corrected for spherical and axial chromatic aberrations see US Patent No.
5,517,360
issued May 14, 1996 to T Suzuki, entitled "Immersion microscope objective."
Changing the design parameters to emphasize, rather than minimize, the
chromatic focal shift can create large chromatic, or hyperchromatic,
aberrations in
the optical path. Such hyperchromatic aberrations can simultaneously focus
multiple
focal depths on a detector, with each optical wavelength forming an image at
the
detector of a separate focal plane within the object. This widens the range of
focal
positions over a limited desired wavelength range. For a specimen with a
narrow
absorption peak in the stain or contrast agent, a lens can be designed to
include
optical field extension elements to extend the dispersion over many microns to
form
an extended depth of field optical system for a narrow range of wavelengths.
The
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optical components and materials are chosen to optimize chromatic dispersion
within
the stain absorption range.
When employing chromatic aberration, it may be advantageous to alter the
relative makeup of the spectral components to compensate for factors that may
affect the composition of the image. These may include, but are not limited
to,
wavelength dependencies of the contrast agent or stain, the camera response,
and
transmission through the optical materials. The spectral composition may be
altered
by, for example, incorporating in the illumination, collection, and/or imaging
optics a
filter that attenuates some wavelengths more than others.
As an example, a limited extension of the depth of field can be achieved for a

narrow range of wavelengths accommodating existing stain absorption curves,
such
as the hematoxylin family of stains. Stains in the hematoxylin family exhibit
a peak
absorption in the wavelength range from 550 to 620 nanometers.
Example 1
One example of a hyperchromatic objective lens 103, suitable for use in a
hyperchromatic system, is depicted in FIG. 1. This compound lens comprises
eight
optical elements 1-8 of which optical elements 1 and are cemented together to
form
a first doublet, and optical elements 3 and 4 are cemented together to form a
second
doublet, 5 and 6 are cemented together to form a third doublet, and 7 and 8
are
cemented together to form a fourth doublet. The first surface of 1 is flat or
slightly
convex, so as to avoid trapping air in a cavity when this surface comes in
contact
with an immersion liquid such as oil or water. An example prescription for the

objective 103 follows.
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Front Radius Back Radius Center
of Curvature of Curvature Thickness
Element Material (mm) (mm) (mm)
(Schott
designation)
1 SF57 200 -3.17 3.54
LAK14 3.17 -5.92 5.08
Air 1
3 KZFSN4 104.5 -6.55 3.64
4 SF6 6.55 -13.77 1
Air 1
SF64 10.73 6.27 4.75
6 LASF40 -6.27 4.47 7.88
Air 2.24
7 5K2 7.23 -3.95 9.05
8 F2 3.95 19.37 8.83
The location of the aperture stop may be chosen to provide telecentricity, and
to
minimize lateral color (also known as chromatic difference of magnification).
Component materials are commercially available from, for example, Schott North

America, Inc. Elmsford, NY 10523.
As shown in the examples described hereinbelow with reference to the
figures, lens system 103, when placed in front of a tube lens having a focal
length of
180 mm, will provide 60X magnification at numerical aperture (NA) equal to 0.9
over
a wavelength range from 550 nm to 620 nm, provided that the space between the
front surface of the first element 1 and the top of a cover slip positioned in
the field of
view of the lens is filled with water. The cover slip is typically about 130
microns
thick, while the water-filled space between the cover slip and the lens may be
about
200 microns thick. An object is focused on the imaging plane of the camera
over a
range of 15 microns at separate wavelengths over a 200-micron diameter field
of
view. In this example embodiment, the portion of the object in a first plane
is focused
by the 550-nm portion of the incident light, a second plane located 5 microns
below
the first plane is focused by the 573-nm portion of the incident light, a
third plane
located 10 microns below the first plane is focused by the 597-nm portion of
the
incident light, and a fourth plane located 15 microns below the first plane is
focused
by the 620-nm portion of the incident light.
System MTFs
Referring now to FIG. 2A through FIG. 2D qualitative examples of simulated
monochromatic modulation transfer functions (MTFs) for each of four
wavelengths,
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as evaluated at four separate image planes. Throughout FIG. 2A ¨ Fig. 3D the
vertical axis represents system MTF and the horizontal axis represents
frequency
ranging from 0 to a cutoff frequency, fc, where fc is the highest frequency
with a non-
zero MTF. An MTF similar to the one shown in FIG. 2A could, in principle, be
measured by placing a 2D optical test target in the object space, illuminating
it with
narrowband light having a wavelength of about 550 nm, and finding the best
focus.
Changing the wavelength to about 573 nm and moving the focal position by 5
microns may produce the MTF shown in FIG. 2B. Repeating this process for about

597 nm and again for about 620 nm yields the MTFs shown in FIG. 2C and FIG.
2D,
respectively. The in-focus information from the entire volume of an object may
be
acquired simultaneously, for an object thickness up to 15 microns.
Referring now to FIG. 3A through FIG. 3D qualitative examples of simulated
polychromatic modulation transfer functions (MTFs) are shown for an optical
projection system where the illumination consists of a band of wavelengths
between
about 550 nm and 620 nm are shown. An MTF similar to the one shown in FIG. 3A
could, in principle, be measured by placing a 2D optical test target in the
object
space, illuminating it with broadband light having a band of wavelengths in
the range
of about 550-620 nm, and finding the best focus, AZ=0. Moving the focal
position by
microns to focus AZ=5 microns qualitatively yields the MTF shown in FIG. 3B.
Moving the focal position by 10 microns, ,62=10 microns, and by 15 microns,
,62=15
microns, qualitatively yields the MTFs shown in FIG. 3C and FIG. 3D
respectively. It
is to be understood that other prescriptions and optical designs may be
employed
without departing from the spirit of this embodiment.
The hyperchromatic optical system may advantageously be incorporated into
an OPTM system. A translation device, such as, for example, a piezoelectric
transducer (PZT) may be used to apply a single, low-speed translation of the
objective lens over the course of a 360-degree set of scans. The lens
translation
keeps the object of interest within a focus interval of about 15 microns, even
while
the tube rotation causes the object to translate along the optical axis by as
much as
the internal diameter of the rotating capillary during the 360-degree scan. In
contrast
to earlier embodiments, a high-speed scan taken at each perspective is no
longer
required. As a result, image acquisition speed is no longer limited by the
speed of
the PZT translation device. In addition, synchronization between the tube
rotation
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and translation motion of the lens no longer needs to be as precise, thereby
reducing
the complexity of the OPTM instrument control system.
Now referring to FIG 4A a detail of object space at one viewing angle in an
optical tomography system incorporating a hyperchromatic optical lens is
schematically shown. A cell 114 lies between a first object plane Z1 and a
second
object plane Z2 inside a microcapillary tube 107. The tube 107 may have, for
example, an inner diameter of 50 microns, and the separation between the first

object plane and the second object plane may be, for example, 15 microns. The
microcapillary tube 107 is preferably filled with an optical matching medium
123
matching the internal index to the tube's index of refraction.
In one example embodiment, an assembly 121 preferably includes the
microcapillary tube 107 placed in a viewing area between a first flat optical
surface
120, which may comprise a standard microscope slide, and a second flat optical

surface 108, which may comprise a standard microscope coverslip. The
interstices
between the tube 107 and the flat surfaces 108, 120 are filled with optical
oil 124, or
an equivalent, having an index of refraction that also substantially matches
those of
the tube 107, the flat surfaces 108, 120, and the optical gel 123. The
assembly 121
can be mounted on a microscope, and an optical immersion fluid 109,
comprising,
for example, oil, water, or air, is placed on the side of the assembly 121
that faces
hyperchromatic optics (as shown in FIG. 4B). The outer diameter of the tube
107
may be, for example about 250 microns, the thickness of the coverslip 108 may
be
about 170 microns, and the thickness of the immersion fluid 109 may be between

about 100 and 300 microns.
Broadband light 130 having wavelengths between a first wavelength Al (e.g.,
Al = about 550 nm) and a second wavelength A2 (e.g., A2 = about 620 nm) is
transmitted into the tube 107 by means of, for example, a condenser lens
system. A
first set of ray paths 105 of light having wavelength Al travel from the first
object
plane Z1 and into the immersion fluid 109. A second set of ray paths 106 of
light
having wavelength A2 travel from the second object plane Z2 and into the
immersion
fluid 109. Although not depicted in FIG 4A, it may be understood that light
having
wavelengths Xn, where An is a wavelength between Al and A2, travel from
intermediate object planes, Zn, located between the first object plane and the
second
object plane, along ray paths similar to 105 and 106.

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With reference to FIG 4B, the operation of this system may be more fully
understood. A chromatic filter 110 and a condenser lens 115 provide
illumination
130F having the desired upper and lower wavelength limits (X1, A2). The
incident
light passes through the tube assembly 121, containing, for example, a
biological cell
114. The ray paths 105 and 106,106, having wavelengths X1 and A2 and beginning

near the cell 114 at object planes Z1 and Z2, respectively, pass through the
immersion fluid 109 and the hyperchromatic objective lens system 103, and are
substantially collimated when they reach the tube lens 111. They then pass
through
the tube lens 111 which may, for example, have a focal length of about 180 mm,
and
achieve focus A on the image plane 104 of a CCD camera 112. The objective lens

103 is mounted on a PZT 113, which is capable of moving the objective 103
further
from the tube lens 111 and closer to the object planes Z1 and Z2.
Light having a wavelength Xn, where the An wavelength is a wavelength
having a value between X1 and A2, will travel from intermediate object planes,
Zn,
located between plane Z1 and plane Z2, along ray paths similar to 105 and 106,
and
also come to a focus on image plane 104. The wavelength of Xn, relative to X1
and
A2, determines where the intermediate object plane is located, relative to
object
planes Z1 and Z2, in order for it to be focused on image plane 104.
Now referring to FIG. 40, the system of FIG. 4A is shown after the
microcapillary tube 107 has rotated, causing the cell 114 to change its
location and
orientation as well. To compensate for this motion, the PZT 113 (as shown, for

example in FIG. 4B) moves the objective lens 103 by an amount sufficient to
cause
the focused light on the image plane 104 to originate from the a second set of
object
planes Z3 and Z4, via ray path 118 from object plane Z3 for light of
wavelength ?1,
and via ray path 119 from object plane Z4 for light of wavelength A2. Those
skilled in
the art and having the benefit of this disclosure will understand that light
having
varying wavelengths An between X1 and A2 will travel from intermediate object
planes, Zn, located between object planes Z3 and Z4 along ray paths between
ray
paths 118 and 119 will also come to a focus on image plane 104. The
wavelengths
Xn, relative to Al and A2, determines where object planes Zn must be located,
relative to Z3 and Z4, in order for it to be focused on image plane 104. FIG.
4D
shows the system detailed in FIG 40, in particular, illustrating that the PZT
113 has
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moved the objective 103 so that the focused image is shifted to focus B on
image
plane 104.
Those skilled in the art and having the benefit of this disclosure will
appreciate
that the system depicted in FIG. 4A-FIG. 4D allows the camera 112 to acquire
an
image similar to the defined pseudo-projection produced by scanning a well-
corrected objective lens over the entire thickness of the cell 114 and further
permits
the acquisition of multiple pseudo-projections from multiple angles, without
the
necessity of moving the objective lens 103 at a high speed via high-frequency
drive
of the PZT scanning mechanism 113.
Another embodiment employs the hyperchromatic optical path described
previously, having an aberration that produces focus over the thickness of the
object
(e.g., 15 microns) for wavelengths within the range of constant absorption by
the
stain. This embodiment further includes a Chromatic Filter Array (CFA) in the
optical
path, preferably located just before the image plane 104. The CFA may consist
of
two or more types of pixels, each pixel having a size corresponding to the
pixel size
of the camera 112 imaging surface 104. Each type of pixel passes a separate
range
of wavelengths. An example of a CFA, having much wider bandpass ranges than
the one described as part of this invention, is the Bayer filter, as described
in U.S.
Patent No. 4,081,277, "Method for making a solid-state color imaging device
having
an integral color filter and the device" issued on 03/28/1978 to Brault, et
al.
Referring now to FIG. 5, an example of a design for a chromatic filter array
is
schematically shown. CFA 201 includes a plurality of pixel types in a unit
cell 202. In
one example, the plurality of pixel types includes four pixel types 203, 204,
205, 206
included in the unit cell 202. The four pixel types pass only wavelengths, of
550-567
nm, 567-584 nm, 584-601 nm, and 601-618 nm, respectively. The CFA 201 must
comprise a sufficient number of unit cells 202 to cover a substantial portion
of the
image surface 104. For example, if the image plane 104 comprises 900 x 1600
pixels, then the CFA 201 may advantageously comprise 450 x 800 unit cells 202,

each unit cell 202 in turn comprising a 2x2 matrix of one each of filter pixel
types
203, 204, 205, and 206. This design may limit the spatial resolution by a
factor of
two, due to the use of only one-fourth of the pixels for each wavelength.
However,
this is in practice not an issue, as long as the pixel-limited resolvable size

(equivalent, with the CFA included, to 4*[pixel size]/magnification) is less
than the
desired optical resolution (in this example, 500 nm). Under typical conditions
of
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camera pixel size = 7.4 microns and magnification = 60, this requirement is
fulfilled.
The light intensity reaching each CCD pixel in this embodiment is reduced by a

factor of four due to the four-wavelength CFA. This reduction is also not a
problem in
practice, as the source intensity can be increased to provide higher light
levels
without requiring longer camera exposure times.
With reference jointly to FIG. 6A and FIG. 6B, another example of the
operation of a hyperchromatic system incorporating a CFA 201 is illustrated.
The
system is similar to the one depicted in FIG. 4A and 40, with the addition of
the CFA
201 on the image surface 104 of the camera 201. Due to translation of the
objective
103 along the optical axis of the system, the focus point shifts from focus
point A' in
FIG. 6A to focus point B' in FIG. 6B.
The inclusion of the CFA 201 makes it possible to separate the signals from
two or more (in this example, four) focal ranges, thereby decreasing the
amount of
defocusing that contaminates the in-focus signal. By saving each focal range
separately, they may be combined digitally during post-acquisition processing,

permitting an increase in the dynamic range of the combined images, and
consequently improving the spatial resolution and contrast of the combined
images.
Alternatively, the images that result from each wavelength can be processed as
two
or more separate sets of data, and not combined until after each has been
separately tomographically reconstructed, thus providing an improvement in
spatial
resolution and contrast.
Referring now jointly to FIG. 7A and FIG. 7B, object space for a first viewing

angle, el, for a hyperchromatic optical tomography system incorporating a long

depth of field is schematically illustrated. The extended limits of the focal
range,
combined with the CFA 201, make a lens transducer, such as PZT 113,
unnecessary
for the operation of the system. Another pair of object planes Z5 and Z6
corresponds to wavelengths X1 and X2, respectively. The object planes are
located
at the extrema of the inner diameter of the microcapillary tube 107. Because
object
planes Z5 and Z6 are located at the extrema of the inner diameter of the
microcapillary tube 107, the location of object planes Z5 and Z6 remain
constant
relative to the objective lens even as the tube 107 rotates, causing the cell
114 to
change its location relatively to the objective lens. For a microcapillary
tube 107
having an inner diameter of 50 microns, the separation between object planes
Z5
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and Z6 should, preferably, be at least 50 microns. The ray paths 405 and 406,
comprising light of wavelengths X1 and X2, respectively, travel through object
planes
Z5 and Z6, respectively, and reach the CFA 201, where X1 is transmitted only
through, for example, CFA pixels of the first type 203, and X2 is transmitted
only
through, for example, CFA pixels of the last type 206.
Owing to the existence of the multiple pixel types in the unit cell of the
CFA,
each pixel type only collects light from a portion of the interval between the
object
planes Z5 and Z6. For the four-color CFA 201 shown in FIG. 5 as described
above,
each interval is, preferably, non-overlapping, and therefore only one-fourth
of the
total interval is transmitted through any pixel type and collected by the
camera 112.
As an example, if the focal plane separation is 50 microns, and the
wavelength range is 550 to 618 nm, then camera pixels lying directly behind
pixel
type 203 will detect only light having wavelengths between 550 and 567 nm,
corresponding to object planes between object plane Z5 and Z5+12.5. In a
similar
manner, camera pixels lying directly behind pixel type 204 will detect only
light
having wavelengths between 567 and 584 nm, corresponding to focal planes
between object planes located between Z5+12.5 microns and Z5+25 microns.
Camera pixels lying directly behind pixel type 205 will detect only light
having
wavelengths between 584 and 601 nm, corresponding to object planes between
Z5+25 microns and Z5+37.5 microns; and camera pixels lying directly behind
pixel
type 206 will detect only light having wavelengths between 601 and 618 nm,
corresponding to object planes between Z5+37.5 microns and Z6 (i.e., Z5+50
microns).
Referring now to FIG. 7B, a first viewing angle for a hyperchromatic optical
tomography system incorporating a long depth of field is schematically
illustrated.
The components comprising the system are similar to those of FIG 6B, except
that a
mechanical translator for the objective lens, such as a PZT, is no longer
necessary.
The ray paths 405 and 406 originate at opposite sides of the tube 205 and
follow
similar paths to the image sensor 104 at focus point A". A CFA 201 is also
shown,
although it is optional in this embodiment.
Referring now to FIG. 7C, there schematically illustrated is the object space
for a second viewing angle for a hyperchromatic optical tomography system
incorporating a long depth of field. Here the cell 114 is rotated to a second
viewing
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angle e2. Because the cell 114 is always within the boundaries of the extended

depth of field (i.e., between planes Z5 and Z6), it is not necessary to employ
a PZT
to move the objective lens 103.
Referring now to FIG. 7D, a second viewing angle for a hyperchromatic
optical tomography system incorporating a long depth of field is schematically

illustrated showing the system detailed in FIG 70. In contrast to FIG. 7B, it
should be
noted that the focus of objective 103 has shifted to focus point B" on image
plane
104. A CFA 201 is also shown, although it is optional in this embodiment.
Referring now to FIG. 8A, an example of multiple camera system where a
capillary tube holding a specimen is at a first rotation angle is shown. A
multiple
camera system includes a chromatic filter 110 and a condenser lens 115, and a
tube
assembly 121, containing, for example, a biological cell 114 substantially as
described hereinabove with reference to FIG. 4A and FIG. 4B. Ray paths
beginning
near the cell 114 at object planes Z1 and Z2, respectively, pass through the
immersion fluid 109 and the hyperchromatic objective lens system 103. In a
departure from the system described above with reference to FIG. 4A and FIG.
4B,
the multiple camera system here incorporates a dichroic beamsplitter cube 501
to
split a first plurality of ray paths 502 and 503. The first plurality of ray
paths 502 and
503 originate in object planes similar to object planes Z1 and Z2. Each camera
may
optionally be filtered by a chromatic filter array 201, 508. In an alternate
embodiment,
a polarization filter array may be substituted for each chromatic filter
array. If a wide
depth of field is desired, then another embodiment, similar to this one, would
employ
CFAs 201 and 508 while eliminating the translational mechanism 113 for moving
the
objective lens similarly to other embodiments described above.
Referring now to FIG. 8B, an example of the multiple camera system of FIG.
8A where the capillary tube holding a specimen is at a second rotation angle
is
shown. Here the dichroic beamsplitter cube 501 splits a second plurality of
ray paths
504 and 505. The second plurality of ray paths 504 and 505 originate in object

planes similar to object planes Z3 and Z4.
Referring now jointly to FIG. 8A and FIG. 8B, the rays 502 and 504, being of
the same wavelength, travel through the first tube lens 111 to the first
camera 112.
The rays 503 and 505, being of a wavelength different from rays 502 and 504,
travel
through the second tube lens 506 to the sensor area 509 of a second camera
507.
Additional dichroic beamsplitters and cameras may be readily envisaged.

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Focus Score
One characteristic of an OPTM system incorporating extended depth of field
optics is that a fixed-focal plane image can no longer be acquired through the

extended depth of field optical path. The focus quality of a flat object is
retained over
a wide range of focal positions. This property is sometimes referred to as
focus
invariance.
For an object that is not confined to a single focal plane, it is still
necessary to
find the midpoint of the object of interest so that it may be kept within the
focus
interval throughout the data acquisition. One method of accomplishing this is
to split
the optical path prior to introducing the chromatic aberration, so that a
separate
optical path, incorporating a detector, is available. This separate optical
path, being
free of chromatic aberration, allows the system to acquire fixed-focal plane
images.
In a similar method that can be incorporated into a hyperchromatic imaging
system,
the optical path can be split and one arm chromatically filtered to near-
monochromaticity, so that a single focal plane can be imaged by a separate
camera,
while the other arm provides the pseudo-projection. Another approach includes
panning the objective lens over a wide range prior to beginning the scan,
acquiring
an image at each position, and assigning a focus score to each image. Focus
scoring methods may employ autocorrelation, entropy, and/or other equivalent
methods.
Referring now to FIG. 9, an example of focus invariance in an MTF range is
illustrated. A focus-invariant range for the MTF at one spatial frequency, MTF
(f1)
601 is bounded on the optical axis by an upper focal plane 602 and a lower
focal
plane 603. Within these boundaries the MTF 601 remains at a roughly constant
plateau value before dropping down to a much lower level. Using such an
approach,
as described further below, the two end-points of the plateau 602, 603 in the
MTF
(f1) 601 can be identified, and the preferred focus chosen by, for example,
the mid-
point 604 between the end-points 602, 603.
Repetitive focus scoring is not necessary for a system having a depth of field

exceeding the inner diameter of a microcapillary tube, provided that the upper
and
lower boundaries 602, 603 of the focus invariance region do not pass through
the
interior of the microcapillary tube. This condition can be verified by an
initial focus
scoring when the instrument is first configured.
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Referring now to FIG. 10, a block diagram of an example method for finding
the midpoint of the focus-invariant region in a focus-invariant optical
tomography
system is schematically shown. According to the example method the midpoint is

found and used to compute a 3D reconstruction of an object by:
1. panning through the optical axis and acquiring multiple images of an object

in a microcapillary tube while panning (701);
2. evaluating the focus quality at each position along the optical axis (702);
3. determining two break points on the optical axis where the focus quality
begins to degrade (703), where the two break points correspond to the
upper and lower boundaries 602, 603 of the focus invariance region;
4. acquiring a pseudo-projection image (704), with the center of the pseudo-
projection's scanning range centered between the upper and lower
boundaries 602, 603 of the focus invariance region;
5. rotating the microcapillary tube to a next projection angle;
6. repeating steps 1-5 until a plurality of pseudo-projections have been
acquired at a plurality of projection angles (705);
7. computing a 3D reconstruction using the acquired pseudo-projections
(706).
Referring now to FIG. 11A, a block diagram of another method for finding the
midpoint of the focus-invariant region in a focus-invariant optical tomography
system
is shown. According to this alternate example method the midpoint is found and

used to compute a 3D reconstruction of an object by:
1. for a first viewing angle, acquiring pseudo-projections at a plurality of
focal
planes 801 by stepping the focus (i.e. by moving the objective lens a short
distance
represented by n=n+1, where n is an incremental step) and acquiring pseudo-
projection data at each focal plane 813;
2. moving to a next viewing angle and repeating the acquisition of pseudo-
projections 801 until a target volume is covered by stepping the focus as
above 802;
3. summing all pseudo-projections for each viewing angle 803 to produce a
set of summed pseudo-projections; and
4. computing a 3D reconstruction using the set of summed pseudo-projections
804.
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In one example, pseudo-projections are acquired until the following formula is
met or
exceeded at 802:
EnD=tube diam, where 0<c<1.
Referring now to FIG. 11B shows a block diagram of yet another method for
finding the midpoint of the focus-invariant region in a focus-invariant
optical
tomography system. According to this alternate example method the midpoint is
found and used to compute a 3D reconstruction of an object by:
1. for a first viewing angle, acquiring pseudo-projections at a plurality of
focal
planes 801 by stepping the focus (i.e. by moving the objective lens a short
distance represented by n=n+1, where n is an incremental step) and acquiring
pseudo-projection data at each focal plane 813;
2. moving to a next viewing angle and repeating the acquisition of pseudo-
projections 801 until a target volume is covered 802 by stepping the focus as
above 802 according to a limiting formula;
4. performing a 2.5-D focus evaluation 805 to determine a best focus pseudo-
projection for each viewing angle; and
5. computing a 3D reconstruction using a set of best focus pseudo-projections
acquired at the best focus for each angle 806.
The method above is similar to that of FIG. 11A except for the 2.5-D focus
evaluation.
Referring now to FIG. 12, an embodiment of an autofocusing system using
chromatic balance is schematically shown. Here two autofocusing cameras 2002,
2004 provide digitized input to an image comparator 2006, which in turn
provides a
feedback signal 2008 to a transducer drive 2010, such as a PZT controller. As
an
object of interest 2001 is rotated within a microcapillary tube (as described
above),
separate images from an upper object volume 2012 and a lower object volume
2014
are captured by the autofocusing cameras 2002, 2004. The images are compared
and analyzed by the comparator 2006. The feedback signal 2008 from the
comparator 2006 drives the transducer drive 2010 which, in turn, controls an
objective lens focus drive 2016 so that the focus range of an objective lens
2103
moves closer to the region of poorer focus quality. When the difference in
focus
quality between the two images becomes sufficiently small, the transducer
drive is
no longer required for shifting the focus range. This process can be repeated
for
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each projection angle, which may be necessary as the planes containing the
object
of interest move up and down the optical axis as the tube rotates. Images are
acquired by an image camera 2020.
In one embodiment, light rays are directed by a primary beam splitter 2028, a
secondary beam splitter 2030 and a mirror 2032. Light rays directed to the
image
camera 2020 are filtered by a first filter 2022, where the first filter passes
light having
wavelengths between 550 nm and 620 nm through a first image forming lens 2023.

Light rays directed to the first autofocusing camera 2002 are filtered by a
second
filter 2024, where the second filter passes light having wavelengths between
585 nm
and 620 nm through a second imaging lens 2025. Light rays impinging mirror
2032
are directed to the second autofocusing camera 2004 after being filtered by a
third
filter 2026, where the third filter passes light having wavelengths between
550 nm
and 585 nm through a third imaging lens 2027.
Referring now to FIG. 13, another embodiment of an autofocusing system
using chromatic balance is schematically shown. A source of spectrally uniform
light
illumination 2060 illuminates an object of interest 2001 which is imaged by
image
camera 2020. Similarly to the autofocusing system described above, light rays
are
directed by a primary beam splitter 2028, a secondary beam splitter 2030 and a

mirror 2032. Light rays directed to the image camera 2020 are filtered by a
first filter
2022, where the first filter passes light having wavelengths between 550 nm
and 620
nm through a first image forming lens 2023. A portion of light rays passing
through
the secondary beam splitter 2028 are filtered by a second filter 2024, where
the
second filter passes light having wavelengths between 585 nm and 620 nm. Light

rays impinging mirror 2032 are filtered by a third filter 2026, where the
third filter
passes light having wavelengths between 550 nm and 585 nm. A first Fourier
plane
forming lens 2050 transmits light from the second filter 2024 through a first
Fourier
plane spatial filter 2052 to a first photo sensor 2054. A second Fourier plane
forming
lens 2056 transmits light from the third filter 2026 through a second Fourier
plane
spatial filter 2058 to a second photo sensor 2054.
The Fourier spatial filters 2052, 2058 operate on two focus paths to provide
analog feedback to the focus control controller 2042 via the photo-diodes
2054.
Spatial filtering ensures that the photodiodes only receive the high-spatial
frequency
components of a focal plane. High spatial frequency content is associated with
well-
focused objects. The high frequency content of the upper and lower halves of
the
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focal range, 2012, 2014 respectively, is compared in signal conditioning and
difference amplification processor 2062. The difference amplification
processor 2062
provides output 2040 which is used as above to control drive 2042 to cause the

transducer 2016 to position the objective lens 2103 until the high-frequency
intensities of the two focal regions are sufficiently similar.
Under continuous
illumination, this method has the advantage of tracking motion of an object
keeping it
in focus balance at all times.
Polarization-dependent optics (birefringent) for 3D Imaging
The location of the focal plane is dependent on the polarization of the light.

This system can be implemented using birefringent optics, in which the index
of
refraction varies according to the electric-field polarization of the optical
wavefront.
An example of a birefringent optical material is calcite (CaCO3), for which
the index
of refraction at 590 nm is either 1.658 or 1.486, depending on the
polarization.
Embodiments analogous to those of the hyperchromatic systems described
above may be employed. With these techniques, the polarization of the imaged
light
will depend on the object focal plane from which it originated. For example,
the
horizontally-polarized (electric-field vector at zero degrees) component of
the light
may provide the in-focus image for an object plane ZH, whereas the vertically-
polarized (electric-field vector at 90 degrees) component of the light may
provide the
in-focus image for an object plane Zv, located, for example, 15 microns closer
to the
detector than plane ZH. Light having polarizations between zero and 90 degrees

would provide in-focus images for object planes between ZH and 4/.
The polarization of the illuminating light can be varied over time by using a
spinning polarizing filter, the collected (unpolarized) light passes through a
polarizing
filter before it reaches the image sensor, or the entire focal range can be
collected
simultaneously.
In one embodiment, the focal range may be comparable to the thickness of
the object, e.g., 15 microns. In this embodiment, a PZT can be incorporated to

compensate for rotation-induced translation of the cell, in a system analogous
to that
depicted in FIG. 6A and FIG. 6B.
In another embodiment, analogous to that depicted in FIG. 7A-FIG. 7D, the
range of the focal planes can be equivalent to the diameter of the
microcapillary tube
(e.g., 50 microns), and a Polarization Filter Array (PFA; the polarization
analog of the

CA 02703102 2010-04-20
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chromatic filter array illustrated in FIG. 5) is incorporated into the system
in place of
the CFA shown in FIG. 7A- FIG. 7D.
In yet another embodiment, the range of the focal planes can be equivalent to
the diameter of the microcapillary tube, and the polarization of the light
varied over
time while a series of synchronized camera exposures acquires the object
planes as
they come into focus on the detector.
2.5-0 imaging
In any OPTM system incorporating extended depth of field optics, post-
acquisition processing may be incorporated to perform pixel-by-pixel analysis
to
compute a mosaic of in-focus features in the field of view. An example of one
type of
2.5-D imaging is found in RJ Pieper and A Korpel, "Image processing for
extended
depth of field," Applied Optics 22, 1449 (1983). The 2.5-D imaging approach
may be
most advantageously employed in those embodiments that make use of a Chromatic

or Polarization Filter Array (CFA or PFA) and covering a wide focal range, and
in the
embodiments that make use of multiple camera exposures. In these systems, the
weight assigned to an element type can vary from one pseudo-projection to the
next,
as the object is rotated through different focal plane regions.
To accomplish this, individual features are identified in the collection of
short-
focal-plane images that form an image stack. The same feature may appear in
several images within the stack, but only a subset of those images will
contain a
well-focused representation of that feature.
Referring now to FIG. 14, a block diagram of a 2.5-D focusing method is
schematically shown. Features, Gi = G1, ... GO in an image stack Sk are
identified
1101. For each feature Gi in an image stack Sk, the images for which Gi is in
best
focus are identified 1102. A blank composite image PPk is generated 1103. The
pixels that make up that best-focused feature (Xi, Yi, Zi) are added to the
composite
image PPk 1104. This process is repeated for all features (G1, G2 ... GO until
all
features have been incorporated into PPk. Since a single feature may span two
or
more images in Sk, a single pixel in PPk may accumulate two or more intensity
values, one for each image in Sk that contains a well-focused representation
of G.
Furthermore, two or more features may share the same X-Y coordinates (but
different Z-coordinates), which may result in some pixels in PPk accumulate
intensity
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values from two or more features. Note that subscripted letters I,k etc.
represent
index numbers.
The process is repeated for all the image stacks, until all image stacks (Si,
S2... Skmax) have been analyzed and their associated composite images (PPi,
PP2...
PPkmax) have been computed 1105. The tomographic reconstruction can then be
computed, using the set of PPk as the input images 1106. In one example using
this
method, each 2x2 block of a 4-color CFA or PFA can be processed by selecting
the
single pixels containing the best focus, or as a weighted sum of two or more
pixels.
Beam split multiple focal plane
There are several fundamental advantages of shorter integrated pseudo-
projections for OPTM performance. First, smaller magnitude pseudo-projections
(integrated optical axis scans) reduce the effect of the low frequency
information
dominating in the spatial spectrum. Second, adding more images that sample the

same volume improves the signal to noise proportionally to the square root of
the
number of images used. Third, multiple images enable the detection and
compensation for unusual hot spots in images due to refractive contrast.
The separation of the depth of field into segments allows many other depth of
field extenders to work to supply a more limited solution, working better with
less
complication.
A reduced range of motion or an extended depth is possible with direct
objective scan and multiple camera focal planes.
The creation of multiple focal ranges does not necessarily require multiple
cameras. With adequate camera sensor area it is possible to merge the images
and
capture them on a single sensor. This can be done using a fiber optic
faceplate
splitting the sensor into zones, or a folded optical system merging the
multiple
images onto a single CCD.
Referring now to FIG. 15, an example of a folded system is depicted. The
substantially collimated light exiting the objective 1205 is divided by a
first dichroic
beam-splitting cube 1206. One arm 1202 has a wavelength Al and originates in a

first focal plane 1201. It passes through a first tube lens, through a second
beam-
splitter cube 1207, and onto the right half of the camera sensor's active area
1211.
The other arm 1204 has a wavelength A2 and originates in a second focal plane
1203. It reflects off two mirrors 1208, 1209, passes through the tube lens
1210,
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CA 02703102 2010-04-20
WO 2009/055429 PCT/US2008/080726
reflects within the second dichroic beam-splitter cube 1207, and onto the left
half of
the camera sensor's active area 1211. The two halves of the camera will
acquire
focused images having substantially identical magnifications, but originating
from
different focal planes 1201, 1203 in object space. The relative lateral shift
in the
images is achieved by laterally shifting the second dichroic beam-splitter
cube 1207,
so that the reflected light of the second arm 1204 is laterally shifted
relative to the
first arm 1202 and to the tube lens 1210.
Two-stage magnification
Acquiring images separated by 10 microns in object space would require, for
a 100x lens, a difference in image-space path length proportional to
magnification
squared (i.e., 100 mm). If the tube lenses have the same focal lengths, but
different
back focal planes, then the two halves of the camera will acquire focused
images
having substantially identical magnifications, but originating from different
focal
planes in object space. As an illustration, placing the camera 100 mm closer
to the
second tube lens than to the first tube lens will result in a difference in
focal planes of
100/M2 microns, where M is the lateral magnification. If M=100, then 100/M2 =
10
microns.
However, a much more modest change in optical axis can be achieved using
two 10x magnification stages and changing the focal plane of the secondary
objective only slightly. A 10-micron shift in the specimen plane at 10x
magnification
image is achieved with a one-millimeter shift of the intermediate image plane.
Using a split focal plane approach allows two or more cameras (four are
shown in the example of FIG. 16) to each collect a range of the focal depth of
the
specimen. In the limit the number of cameras is practically limited by the
amount of
light that can be brought to illuminate the specimen and the cost and
complexity of
the optical path. A system incorporating more cameras improves signal to
noise,
assuming that the cameras' well capacities are sufficiently close to full, and
reduces
the range that each image must deliver in field depth. A shorter field depth
aids in
producing better representation of high spatial frequencies in a resultant
pseudo-
projection.
In an example of this embodiment, shown in FIG. 16, an optical system
comprises a primary objective 1302, first and second mirrors 1304A, 1304B,
three
beam-splitters 1303A-13030, four primary tube lenses 1310, four secondary
23

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WO 2009/055429 PCT/US2008/080726
objective lenses 1312, four secondary tube lenses 1308, and four CCD cameras
1309. The primary objective 1302 and the primary tube lenses 1310 provide at
least
10X magnification, and the secondary objectives and tube lenses provide at
least an
additional 10x magnification, for a total of at least 100x magnification.
Each ray path passes through two beam-splitter thicknesses, and each ray
path undergoes either two or zero reflections, either through the beam-
splitters or by
the mirrors. The equivalence of the ray path-lengths through the beam-
splitters
means that the aberrations due to passing through the glass are equivalent.
The
number of reflections being always even (or always odd) means that all four
images
retain the same orientation at the image planes of the four cameras 1309.
Space
between first tube lens and secondary objective differs for each ray path, so
that a
different object plane is focused on each camera. A reduced range of motion or
an
extended depth is possible with focal plane scanning behind the objective and
multiple camera focal planes.
Extending the multiple camera optics to greater than 20 focal planes can, in
theory, sample a ten-micron depth of field every 500 nm. The arrangement of
multiple cameras allows two simultaneous modalities of volumetric sampling
that can
each be used to contribute their relative strengths to a more accurate
volumetric
reconstruction. Specifically, the contrast generated by refractive and
diffractive
effects in the sample media interfaces may be sorted out from the purely
absorptive
effects and all data captured rapidly and without focal plane motion or
rotational blur.
Wavefront coded optics
Referring now to FIG. 17, a schematic diagram of an OPTM system including
wavefront coded optics is shown. As above, a microcapillary tube 1707 holds an

object of interest 1701 and is rotated through various viewing angles as
viewed by
an objective lens 1703. Light transmitted through the objective lens 1703
impinges
on wavefront coded optics 1705 which are located between the objective lens
1703
and an imaging camera 1707. The use of wavefront coded optics provides a
method
of pre-distorting the optical wavefront so that an object of interest is
contained within
an extended depth of field producing a low but consistent frequency response
throughout its volume. Thus all focal planes, within a limited range along the
optical
axis, are equally defocused. This constitutes the wavefront coding. Wavefront
coding elements are available from CDM Optics, Inc. (Boulder, CO), and are
24

CA 02703102 2010-04-20
WO 2009/055429 PCT/US2008/080726
described in, for example, ER Dowski, "Wavefront coding optics," US Patent No.

6,842,297 (2005).
The limit of wavefront coding is about a 12:1 improvement in the depth of
field. For an optical tomography application such an improvement will provide
about
half of the required depth. Thus wavefront coding may advantageously be
combined
with one of the many other embodiments described herein to deliver a complete
solution.
The point of the first contrast-reversal (MTF less than zero) occurs, for
matched condenser and objective NA's, at 0.64 waves of defocus, as detailed in
VN
Mahajan, "Aberration Theory Made Simple" (Bellingham, WA: SPIE Press, 1991).
This point is readily expressed in terms of the change in the optical depth,
Az, as
Az = 1.28Xn/(NAobi)2
where X is the wavelength of the light being collected, n is the refractive
index of the
region between the objective lens and the object, and NA0bi is the numerical
aperture
of the objective lens. For X =550 nm, n = 1, and NA0bi = 0.9, this distance is
Az =
0.87 microns. Then for a 12-micron-deep object, we require at least a 5x
improvement in the depth of field to avoid contrast reversal at 6-micron
defocus
(roughly 4.4 waves of defocus).
Another embodiment of imaging with wavefront coding incorporates digital
enhancement of the image with a complementary transfer function to boost the
suppressed high frequency components to recover a sharply focused image while
retaining the extended depth.
Another embodiment uses multiple cameras, such as is shown above, that
take advantage of the wavefront coded optics approach to extended depth of
field by
coding each optical path with lens transfer function, thus extending the depth
of field
from one segment to the next. This mechanism allows for a single brief
exposure
such as a strobed illuminator to quickly sample a wide depth of field without
mechanical motion.
The invention has been described herein in considerable detail in order to
comply with the Patent Statutes and to provide those skilled in the art with
the
information needed to apply the novel principles of the present invention, and
to
construct and use such exemplary and specialized components as are required.
However, it is to be understood that the invention may be carried out by
specifically

CA 02703102 2015-10-19
77501-46
different equipment, and devices, and that various modifications, both as to
the
equipment details and operating procedures, may be accomplished without
departing from the true scope of the present invention.
26

Representative Drawing
A single figure which represents the drawing illustrating the invention.
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Title Date
Forecasted Issue Date 2016-11-29
(86) PCT Filing Date 2008-10-22
(87) PCT Publication Date 2009-04-30
(85) National Entry 2010-04-20
Examination Requested 2013-10-04
(45) Issued 2016-11-29

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Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $400.00 2010-04-20
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Reinstatement: Failure to Pay Application Maintenance Fees $200.00 2013-09-25
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Request for Examination $800.00 2013-10-04
Maintenance Fee - Application - New Act 6 2014-10-22 $200.00 2014-10-09
Maintenance Fee - Application - New Act 7 2015-10-22 $200.00 2015-10-08
Final Fee $300.00 2016-10-12
Maintenance Fee - Application - New Act 8 2016-10-24 $200.00 2016-10-12
Maintenance Fee - Patent - New Act 9 2017-10-23 $200.00 2017-09-27
Maintenance Fee - Patent - New Act 10 2018-10-22 $250.00 2018-10-04
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Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
VISIONGATE, INC.
Past Owners on Record
HAYENGA, JON W.
RAHN, RICHARD J.
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Office Letter 2020-11-02 1 175
Maintenance Fee Payment 2020-10-22 1 33
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Abstract 2010-04-20 1 66
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Description 2010-04-20 26 1,352
Representative Drawing 2010-04-20 1 18
Cover Page 2010-06-11 2 50
Description 2015-10-19 29 1,453
Claims 2015-10-19 11 374
Representative Drawing 2016-11-16 1 10
Cover Page 2016-11-16 1 45
PCT Correspondence 2017-07-27 5 204
PCT 2010-04-20 4 185
Assignment 2010-04-20 2 64
Correspondence 2010-06-08 1 18
Correspondence 2011-01-31 2 135
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Prosecution-Amendment 2015-04-30 3 227
Change to the Method of Correspondence 2015-01-15 45 1,704
Amendment 2015-10-19 19 678
Final Fee 2016-10-12 2 73
Assignment 2016-12-19 3 130